Mycobacterium tuberculosis (Mtb) is the causative agent of what remains one of the most insidious and invasive of human infections, responsible for up to 2 million deaths per year. For most of the course of disease the basic unit of infection is the infected macrophage, therefore the interplay between these two cell types plays a crucial role in determining the outcome of infection. The project details the identification of host environmental cues and bacterial responses that enable the bacterium to invade and survive within the host cell. Genes up-regulated during infection will be exploited to construct fluorescent reporter Mtb strains that express GFP under defined conditions. These reporter strains will be used for both genetic and chemical screens to dissect out the signaling networks and to identify small molecular inhibitors that will form the foundation of a novel drug discovery program. The central hypothesis is that Mtb relies on environmental cues inside its host cell to activate genetic networks to enable it to adapt and survive, and that genetic or chemical interference with these networks will lead to death of the infecting bacteria. The Specific Aims are as follows: 1. Identification of genetic networks activated by intracellular infection. Focuses on the identification of intracellular cues that trigger transcriptional activity in Mtb and the construction of reporter strains that express GFP under the regulation of promoters from those genes up- regulated in response to the environmental cue. 2. Exploitation of genetic screens to identify and delineate regulatory networks. These synthetic phenotypes will be utilized to screen for mutants with aberrant regulation of GFP expression. Such a screen will encompass the sensor/regulator machinery including transcriptional control and feedback loops. The phenotypes of these mutants will be probed in tissue culture and animal models of infection to identify those networks most critical to infection of the host. 3. Exploitation of chemical screens to identify small molecule inhibitors of these networks. High-throughput screens will be conducted on the reporter strains to identify small molecule inhibitors that modulate GFP expression. These hits will be analyzed for their activity against Mtb in macrophages and, in collaboration with Vertex Pharmaceuticals, used as lead compounds for novel target identification and drug development. PUBLIC HEALTH RELEVANCE: Tuberculosis remains a serious global problem with the majority of deaths in poor countries. We have no effective vaccines and constant problems with emerging drug resistance. The central premise of this current proposal is that Mtb up-regulates genetic networks to support intracellular survival and that genetic and chemical screens against these sensor/regulator networks will lead to the identification of novel drug targets and lead compounds.